EP3445384B1 - Zusammensetzung zur behandlung von diastolischer dysfunktion - Google Patents

Zusammensetzung zur behandlung von diastolischer dysfunktion Download PDF

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EP3445384B1
EP3445384B1 EP17727399.2A EP17727399A EP3445384B1 EP 3445384 B1 EP3445384 B1 EP 3445384B1 EP 17727399 A EP17727399 A EP 17727399A EP 3445384 B1 EP3445384 B1 EP 3445384B1
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cardiomyocytes
protein
crystallin
alpha
diastolic
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French (fr)
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EP3445384C0 (de
EP3445384A1 (de
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Walter Joseph PAULUS
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Amsterdam UMC Foundation
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Vrije Universiteit Medisch Centrum VUMC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention in general relates to the field of medicine and particularly to medicaments and therapies for the treatment of diastolic heart failure (DHF) and diastolic dysfunction in a subject. More particularly the present invention relates to the use of a protein encoded by a crystalline alpha B ( CRYAB ) gene in cardiomyocytes of the subject for treating diastolic dysfunction.
  • DHF diastolic heart failure
  • CRYAB crystalline alpha B
  • Diastolic dysfunction and diastolic heart failure refers to the decline in performance of at least the left ventricle of the heart during diastole.
  • Diastole is the cardiac cycle phase during which the heart is relaxing and filling with incoming blood that is being returned from the body.
  • Diastolic dysfunction is considered a condition in which abnormalities in mechanical function of the heart, particularly in the functioning of the ventricles of the heart, are present during diastole. Abnormalities in diastolic function can occur in the presence or absence of a clinical syndrome of heart failure.
  • Diastolic dysfunction appears when the left ventricle isn't filling properly during diastole because the ventricular wall can't relax or because the wall is too thick or rigid. Accordingly any condition or process that leads to stiffening of the left ventricular wall can lead to diastolic dysfunction.
  • Cardiomyocytes or cardiac muscle cells are the muscle cells that make up the cardiac muscle or heart.
  • Each cardiomyocyte cell contains myofibrils arranged in sarcomeres, the fundamental contractile units of muscle cells.
  • Sarcomeres are mainly composed of the protein myosin, which forms a thick filament, and the protein actin, which forms a thin filament, which long, fibrous filaments can slide past each in order to allow the cardiac muscle to contract or relax.
  • the giant cytoskeletal protein titin is an important protein in sarcomeres, which functions as a molecular spring which is responsible for the passive elasticity of the muscle. Titin binds to the thick filament (myosin) system, and provides binding sites for numerous proteins. Stiffening of the cardiomyocytes causing diastolic dysfunction is mainly due to altered elastic properties of titin in these cells.
  • a subject may be determined to have diastolic heart failure if he has signs and symptoms of heart failure, a normal left ventricular ejection fraction and evidence of diastolic left ventricular dysfunction. Whether a subject suffers from diastolic dysfunction can for instance be determined by a Doppler echocardiographic examination of the subject.
  • DHF is a clinical syndrome characterized by an abnormal diastolic function (diastolic dysfunction) combined with symptoms and signs of heart failure and a preserved ejection fraction (EF).
  • diastolic dysfunction diastolic dysfunction
  • EF preserved ejection fraction
  • HEPF preserved ejection fraction
  • contractile performance of the heart is relatively preserved but diastolic left ventricular (LV) function is greatly impaired with slow LV relaxation and high diastolic LV stiffness. No single therapy was so far able to improve diastolic LV dysfunction.
  • diastolic LV dysfunction being the main cardiac disturbance in DHF and no single therapy has so far been shown to alter outcome or symptoms in large randomized trials targeting DHF.
  • diastolic LV dysfunction being the main cardiac disturbance in DHF and no single therapy has so far been shown to alter outcome or symptoms in large randomized trials targeting DHF.
  • a consensus statement on the diagnosis of heart failure with normal left ventricle ejection fraction is provided in Paulus et al (2007). European Heart Journal; Vol 28; pp 2539-2550 .
  • the present invention thus aims among others to provide compositions which are useful for treating diastolic dysfunction and/or diastolic heart failure in a subject, and in particular for use in treatment of diastolic dysfunction in a subject caused by stiffening of the cardiomyocytes in the heart of the subject.
  • the invention is set out in the appended set of claims.
  • the invention provides compositions for use in treatment of diastolic dysfunction and DHF as defined in the claims.
  • the invention provides compositions for use in treating diastolic dysfunction and DHF caused by stiffening of cardiomyocytes in the heart of the subject.
  • the invention provides compositions for use in treatment of a subject with a failing heart demonstrating diastolic dysfunction.
  • the compositions for use according to the invention are particularly capable of reducing or removing increased stiffness of cardiomyocytes in the heart of the subject.
  • the invention provides compositions for use in treatment of diastolic dysfunction and DHF which compositions are capable of restoring the elastic properties of titin in cardiomyocytes of the subject.
  • compositions according to the invention may thus be used to treat subjects which have been determined to comprise stiffened cardiomyocytes, and/or have been shown to demonstrate diastolic dysfunction and/or have been diagnosed to suffer from DHF.
  • subjects that have an amount of stiffened cardiomyocytes in the heart exceeding that of a generally healthy heart, i.e. a heart with a normal diastolic function may be treated with the compositions for use according to the invention. Therefore, apart from subjects with DHF, subjects with other conditions such as aortic stenosis and/or dilated cardiomyopathy may also be treated with the compositions for use according to the invention.
  • Alpha-crystallin B chain also referred to as ⁇ -B crystallin or crystallin alpha B, which is encoded by the CRYAB gene in humans, is part of the small heat shock protein family and functions as a molecular chaperone that primarily binds misfolded proteins to prevent protein aggregation, as well as inhibit apoptosis and contribute to intracellular architecture.
  • External Ids are HGNC: 2389; Entrez Gene: 1410; Ensembl: ENSG00000109846; OMIM: 123590 and UniProtKB: P02511. Post-translational modifications decrease the ability of alpha-crystallin B chain to chaperone.
  • the invention provides a composition for use in treatment of diastolic dysfunction and DHF, which composition comprises a therapeutically effective amount of a substance that increases an activity and/or expression of alpha-crystallin B chain protein in cardiomyocytes of a subject.
  • the substance increases an alpha-crystallin B chain protein level in cardiomyocytes of the subject.
  • the substance may comprise a therapeutically effective amount of a polypeptide having an amino acid sequence of SEQ ID NO: 1 ( figure 7 ) or an amino acid sequence that has an amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 100% with the alpha-crystallin B chain protein of SEQ ID NO: 1.
  • the polypeptide reduces a stiffness of cardiomyocytes in a heart of a subject. More preferably the polypeptide restores the elastic properties of titin in cardiomyocytes of a subject with diastolic dysfunction.
  • the substance provides for enhanced expression of the CRYAB gene or the substance provides a nucleic acid segment encoding a polypeptide that has an amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 100% with the alpha-crystallin B chain protein of SEQ ID NO:1, and particularly having at most a two amino acids difference with the sequence of SEQ ID NO:1.
  • the enhanced expression of the CRYAB gene or the nucleic acid supplements the naturally occurring expression of alpha-crystallin B chain, thereby increasing a total amount or level of the alpha-crystallin B chain protein or functionally equivalent polypeptide in the cell.
  • the enhanced expression may comprise an ectopic expression of the CRYAB gene.
  • Ectopic expression is referred to herein as the expression of a coding sequence in an abnormal place in an organism or from an abnormal chromosomal position in the cell, and/or a sequence coding for a heterologous protein that is not normally expressed in the cell.
  • Ectopic expression is typically achieved by artificially introducing a nucleic acid that codes for the respective expressed nucleic acid in the cell.
  • Ectopic expression of a nucleic acid can be done by introducing a transgene with a (modified) promoter into the target cell (transient or stable transfection).
  • the substance in the composition for use according to the invention increases an alpha-crystallin B chain protein level by enhancing expression of the CRYAB gene in cardiomyocytes of the subject.
  • the substance can for instance comprise a small molecule or small RNA that is capable of increasing CRYAB gene expression in cells of the subject, such as for instance by activating a transcription factor.
  • the substance may alternatively or in addition to such a small molecule or small RNA also comprise one or more other gene regulation peptides that promote gene transcription, such as for example a coactivator, chromatin remodeler, histone acetylase, kinase, and methylase.
  • GGA Geranyl-geranyl acetone
  • GGA is manufactured by Eisai Co., Ltd. under the product name "Selbex”.
  • GGA has the generic name of teprenone, and is widely used as a drug to treat stomach ulcers and stomach inflammation.
  • GGA may be acquired as a reagent or industrial raw material. It can be synthesized using well-known methods of synthesis.
  • the chemical name of GGA is 6, 10, 14, 18-tetramethyl-5, 9, 13, 17-nonadecatetraen-2-one.
  • GGA, or a derivative thereof, particularly the derivative NYK9354 is capable of inducing expression of CRYAB in cardiomyocytes.
  • Administration of an effective amount of GGA or a derivative thereof such as NYK9354 results in higher expression levels of alpha B-crystallin protein in cardiomycocytes. Suitable derivatives and the synthesis thereof are described in US patent 4,169,157 .
  • GGA is a registered drug and as such used in clinical trial and a clinical setting.
  • the total amount of GGA administered over a fourteen-hour period can be, for example, 10-1,000 mg, e.g., 50-500 mg, e.g., 100-300 mg, as determined to be appropriate by those of skill in this art.
  • the amount of GGA administered can vary, depending on the body weight of the patient and/or the tolerance of the patient.
  • the invention also includes the use of GGA in the preparation of medicaments for preventing and treating the diseases and conditions described herein.
  • GGA can be given to individuals at a dose of between 1 mg - 1 gram per day.
  • the drug is preferably administered 3 times a day.
  • Suitable dosage form comprise between 0,33 mg - 1 gram GGA.
  • the dosage form preferably comprises 1-300 mg GGA, preferably 3-100 mg, more preferably 20-80 mg GGA.
  • Clinical trials with GGA are described among others in Hongo et al (2012) J. of Gastroenterology and hepatology; Volume 27; Pages 62-68 ; at clinicaltrials.gov in a study entitled “Efficacy and Safety of Teprenone in Patients With Acute Gastritis, Acute Gastric Lesion of Chronic Gastritis With Acute Exacerbation or Gastric Ulcer”; and a study entitled “The purpose of this study is to evaluate the efficacy of teprenone on chronic non-atrophic erosive gastritis and its therapeutic mechanism".
  • the dosage form is typically an oral dosage form.
  • the enhanced expression of the CRYAB gene in cardiomyocytes of a subject with diastolic dysfunction reduces an existing cardiomyocytes stiffness and therefore is a suitable therapeutic method for treating diastolic dysfunction and the symptoms affiliated therewith in diastolic heart failure.
  • the substance in the composition for use comprises GGA or a derivative thereof, particularly NYK9354.
  • composition for use according to the invention in an alternative embodiment comprises a substance which increases the alpha-crystallin B chain protein activity, for instance a substance that affects a post-translational modification of alpha-crystallin B chain protein in cardiomyocytes of the subject.
  • composition for use according to the invention comprises a substance that mediates a post-translational modification of alpha-crystallin B chain protein to increase the ability of alpha-crystallin B chain to chaperone.
  • composition for use in treating diastolic dysfunction in a subject comprises one or more of the foregoing substances which increase a level of alpha B-crystallin protein in muscle cells, particularly in cardiomyocytes.
  • compositions comprising both an amount of a polypeptide that has an amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 100% with the alpha B-crystallin protein, as well as an effective amount of GGA to induce expression of the CRYAB gene in cardiomyocytes of the subject to further increase an expression level of alpha-crystallin B chain protein in the cardiomyocytes may be used in treating diastolic dysfunction in a subject. Any other suitable combination of two or more of the substances mentioned herein may be taken to prepare the composition for use according to the present invention.
  • alpha-crystallin B chain protein comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having an amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 100% with the amino acid sequence of SEQ ID NO: 1.
  • the protein has at most a two amino acids difference with the amino acid sequence of SEQ ID NO:1.
  • Alpha B chain crystalline protein is a protein of SEQ ID NO: 1 or an orthologue thereof of another species, preferably another mammal alpha B chain crystalline protein which is also an amino acid sequence capable of reducing diastolic stiffness in cardiomyocyte cells of a subject that has a heart demonstrating diastolic dysfunction.
  • the protein affects a stiffness of cardiomyocytes of a subject with diastolic dysfunction.
  • the protein preferably affects the elastic properties of titin in cardiomyocytes of the subject.
  • a CRYAB gene reference is made to a nucleic acid that encodes a protein as defined in this paragraph.
  • nucleic acid shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof.
  • the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof.
  • the CRYAB gene comprises the coding region present in the sequence of SEQ ID NO: 2 ( figure 8 ).
  • the reference may be to the gene as present on the chromosome of a human cell, i.e. including the promoter and transcription signals present thereon and including the introns.
  • the reference also includes a cDNA of CRYAB. It typically refers to the coding region of CRYAB with or without one or more (artificial) introns, such as is typically used in nucleic acid molecules designed for directing heterologous expression of CRYAB.
  • the level of an alpha B chain crystalline protein is increased in a cell when the amount of alpha B chain crystalline protein in the cell is increased by at least 10%, preferably at least 20%, more preferably at least 30, 40, 50% or more when compared to the amount of alpha B chain crystalline protein in the cell prior to said increase.
  • the activity of alpha B chain crystalline protein is increased in a cell when the activity of alpha B chain crystalline protein is increased by at least 10%, preferably at least 20%, more preferably at least 30, 40, 50% or more when compared to the activity of alpha B chain crystalline protein in the cell prior to said increase.
  • Alpha B chain crystalline protein activity in a cell can be increased with or without a concomitant increase in the level of alpha B chain crystalline protein.
  • a preferred but not limiting way of increasing the activity of alpha B chain crystalline protein in a cell is by administration of an effective amount of a substance that increases expression of a heat shock protein in the cell, such as geranylgeranylacetone (GGA) or NYK9354.
  • GGA geranylgeranylacetone
  • NYK9354 geranylgeranylacetone
  • the CRYAB gene or the nucleic acid segment can be encoded by an expression cassette which is provided to said cell.
  • the expression cassette encodes an RNA transcript in which the coding region for the alpha-crystallin B chain protein is contained.
  • the expression cassette in addition to the sequence of interest, i.e. the CRYAB gene or the nucleic acid segment, contains additional sequences, for instance a suitable promoter and a transcription termination sequence, for controlled and/or proper expression of the sequence of interest. It is within the skill of the artisan to design suitable expression cassettes and transcripts.
  • the expression cassette used is preferably a vector, preferably an expression vector.
  • a vector typically comprises a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.
  • a vector containing foreign DNA is termed recombinant DNA.
  • Some types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • the substance comprises a vector which is suitable for transfecting or transducing cardiomyocytes of the subject and which vector carries at least one copy of a nucleic acid encoding CRYAB.
  • the nucleic acid is a nucleic acid comprising the coding region of SEQ ID NO:2 or the nucleic acid segment.
  • the number of copies of the gene or nucleic acid segments used may be adapted to an envisaged amount of alpha-crystallin B chain protein or functionally equivalent polypeptide to be expressed in the cells of the subject.
  • the expression vector is preferably suitable as a gene delivery vehicle to be introduced into the cell.
  • the CRYAB gene or the nucleic acid segment is encoded by a viral or virus-based gene delivery vehicle.
  • a preferred embodiment of the delivery vehicle is a viral vector such as an adenoviral vector and more preferably an adeno-associated virus vector.
  • the substance in the composition according to the invention comprises an adeno-associated virus vector or a lentivirus vector, more particularly a lentivirus vector carrying the CRYAB gene.
  • the invention thus also provides such expression vectors and delivery vehicles carrying the CRYAB gene or a nucleic acid segment encoding a polypeptide that has an amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 100% with the alpha-crystallin B chain protein.
  • An expression cassette, vector or gene delivery vehicle such as a viral vector typically comprises all of the sequences that are required in cis to allow efficient transcription and translation of the coding region.
  • Polypeptides or polynucleotides can serve as the active ingredient in pharmaceutical compositions formulated for the treatment of the disorders as described above, i.e. diastolic dysfunction and/or diastolic heart failure.
  • the active ingredient is present in a therapeutically effective amount, i.e., an amount sufficient when administered to substantially modulate the effect of the targeted protein or polypeptide to treat a disease or medical condition mediated thereby.
  • terapéuticaally effective amount refers to the quantity of the alpha B chain crystalline protein or nucleic acid according to the invention necessary to reduce or diminish an increased stiffening of cardiomyocytes. Amounts effective to achieve this goal will, of course, depend on the type and severity of the disease and the general condition, particularly the weight, of the patient.
  • compositions can also include various other agents to enhance delivery and efficacy, e.g. to enhance delivery and stability of the active ingredients.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • pharmaceutically acceptable carrier refers to a carrier for administration of the active substance.
  • the pharmaceutically acceptable carrier can comprise any substance or vehicle suitable for delivering the substance to a therapeutic target, such as muscle cells, in particularly heart muscle cells, more particularly cardiomyocytes of the subject.
  • the term refers to any pharmaceutical carrier that may be administered without undue toxicity.
  • Suitable carriers may be one or more optional stabilizers, diluents, or excipients.
  • the diluent is particularly selected so as not to affect the biological activity of the combination.
  • examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, and dextrose solution.
  • the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, non-immunogenic stabilizers, excipients and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
  • the composition can also include any of a variety of stabilizing agents, such as an antioxidant.
  • the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake).
  • modifications or complexing agents include sulfate, gluconate, citrate and phosphate.
  • the polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions, and lipids.
  • the pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments, particularly therapeutic treatments.
  • Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for human subjects or patients.
  • the dosage of the active ingredient typically lies within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • compositions described herein can be administered in a variety of different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, or intracranial method.
  • the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbon
  • Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of time, e.g. over several hours.
  • Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • the active ingredient can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane and nitrogen.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions intended for in vivo use are preferably sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is preferably substantially free of any potentially toxic agents, such as any endotoxins, which may be present during the synthesis or purification process.
  • compositions for parental administration are also preferably sterile, substantially isotonic and made under GMP conditions.
  • the alpha-crystallin B chain compositions may be administered in a single dose, or in multiple doses, usually multiple doses over a period of time, e.g. daily, every-other day, weekly, semiweekly, monthly etc. for a period of time sufficient to reduce severity of the disorder or disease, which may comprise 1, 2, 3, 4, 6, 10, or more doses.
  • Determining a therapeutically effective amount of an agent that provides alpha-crystallin B chain activity can be done based on animal data using routine computational methods.
  • the therapeutically effective amount contains between about 0.1 mg and about 1g of nucleic acid or protein, as applicable.
  • the effective amount contains between about 1 mg and about 100 mg of nucleic acid or protein, as applicable.
  • the effective amount contains between about 10 mg and about 50 mg of the nucleic acid or protein, as applicable.
  • the effective dose will depend at least in part on the route of administration.
  • the agents may be administered orally, in an aerosol spray, or by injection.
  • the dose may be from about 0.1 pg/kg patient weight; about 1 pg/kg; about 10 ⁇ g/kg; to about 100 ⁇ g/kg.
  • the alpha-crystallin B chain compositions are administered in a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to an excipient acceptable for use in the pharmaceutical and veterinary arts, which is not toxic or otherwise inacceptable.
  • concentration of alpha-crystallin B chain compositions of the invention in the pharmaceutical formulations can vary widely, i.e. from less than about 0.1 %, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • Treating, treatment, or therapy of a disease or disorder shall mean slowing, stopping or reversing the disease's progression by administration of an alpha-crystallin B chain composition.
  • treating a disease means reversing the disease's progression, ideally to the point of eliminating the disease itself.
  • Preventing, prophylaxis or prevention of a disease or disorder as used in the context of this invention refers to the administration of an alpha-crystallin B chain composition to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.
  • DCM diac cardiomyopathy
  • a portion of the myocardium, the left ventricle of the subject is dilated and left and/or right ventricular systolic pump function of the heart is impaired, leading to progressive cardiac enlargement and hypertrophy.
  • DCM may be diagnosed in a subject when a generalized enlargement of the heart is seen upon normal chest X-ray and echocardiogram.
  • the electrocardiogram often shows sinus tachycardia or atrial fibrillation, ventricular arrhythmias, left atrial enlargement, and sometimes intraventricular conduction defects and low voltage.
  • Cardiac magnetic resonance imaging may also provide helpful diagnostic information in patients with dilated cardiomyopathy.
  • aortic stenosis or "AS” as used herein shall mean the condition in a subject in which the exit of the left ventricle or aortic valve of the heart narrows such that problems, e.g. heart failure, occur.
  • Aortic stenosis may be diagnosed by routine examination of the heart and confirmed by echocardiography. In particular, there may be a slow and/or sustained upstroke of the arterial pulse, and the pulse may be of low volume.
  • subject or "patient” as used herein shall mean any animal, such as a non-human primate, mouse, rat, guinea pig or rabbit, and particularly a human.
  • FIG. 1 F passive in myocardial strips. At baseline, F passive was significantly higher in dilated cardiomyopathy (DCM) strips and aortic stenosis (AS) strips compared to donor (A). After extracting thick and thin filaments, the remaining F passive can be attributed to the E-matrix and this was higher in DCM and AS than in donor (B). * P ⁇ 0.05 DCM vs Donor; # P ⁇ 0.05 AS vs Donor.
  • DCM dilated cardiomyopathy
  • AS aortic stenosis
  • FIG. 2 F passive caused by the E-matrix. AS strips had significantly higher E-matrix based F passive than donor in stretches exceeding 15% from slack length (A). At all stages of muscle strain, F passive caused by the E-matrix was higher in DCM strips than in donor (B). # P ⁇ 0.05 AS vs Donor; * P ⁇ 0.05 DCM vs Donor.
  • FIG. 3 F passive caused by titin.
  • titin-based F passive was higher than in donor strips at 10-20% stretches, but not at the highest stretches (A).
  • F passive caused by titin was higher in DCM strips than in donor (B).
  • Figure 4 F passive in single myocytes.
  • FIG. 5 Effects of Acidosis and Prestretch on F passive in single AS myocytes. Incubating single AS cardiomyocytes in pH 6.6 did not change F passive (A).
  • FIG. 6 Effects of a-B crystallin at Baseline and after Acidosis and Prestretch on F passive in single AS cardiomyocytes. Incubation with ⁇ -B crystallin decreased F passive significantly (A). In single AS cardiomyocytes that underwent prestretch alone (B) or a prestretch in combination with pH 6.6 (C), ⁇ -B crystallin decreased F passive significantly as well. * P ⁇ 0.05 vs AS Baseline.
  • Figure 7 The amino acid sequence (SEQ ID NO.:1) corresponding to a full length human alpha-crystallin B chain protein (CRYAB), natural variants of this protein exist.
  • Figure 8 The nucleic acid sequence (SEQ ID NO.:2) corresponding to a full length CRYAB cDNA of a human CRYAB.
  • FIG. 9 Confocal laser microscopy for ⁇ -B crystallin in donor and aortic stenosis (AS). Confocal laser microscopic images were obtained from left ventricular (LV) myocardium of donor and AS patients with immunohistochemical visualization of cell membranes (A), nuclei (B), and ⁇ -B crystallin (C). In myocardium of AS patients, intensity of ⁇ -B crystallin expression (C) was higher than in donor with visualization in the merged images (D) of subsarcolemmal aggresomes especially in the vicinity of capillaries (white arrows). The latter suggests insufficient covering by ⁇ -B crystalline of sarcomeric proteins like titin in AS.
  • Figure 10 Diagram showing respective responses of donor and aortic stenosis (AS)/dilated cardiomyopathy (DCM) cardiomyocytes to administration of alkaline phosphatase, prestretch, and ⁇ -B crystallin.
  • AS donor and aortic stenosis
  • DCM diilated cardiomyopathy
  • each muscle strip was transferred from relaxing to maximally activating solution (pCa4.5), at which isometric force developed. After stabilization for 5 minutes in relaxing solution, strips were stretched to 10, 15, 20 and 25% relative to slack length and F passive was measured at each stage of muscle strain. Subsequently, thick and thin filaments were extracted by immersing the preparation in relaxing solution containing 0.6 mol/L KCl (45 minutes at 20°C) followed by relaxing solution containing 1.0 mol/L KI (45 minutes at 20°C) 1-3 . After the extraction procedure, the muscle bundles were stretched again and the F passive remaining after KCl/KI treatment was ascribed to the extracellular matrix (E-matrix). At each stage of muscle strain, F passive following extraction was subtracted from baseline value to yield F passive attributable to cardiomyocyte-stiffness, or titin.
  • E-matrix extracellular matrix
  • F passive related to titin cannot be explained by isoform shifts because both DCM and AS express more of the compliant N2BA titin isoform.
  • F passive related to titin we subjected isolated cardiomyocytes to treatment with AP to discern the contribution of phosphorylation and to conditions prevailing in failing myocardium such as high stretch and hypoperfusion-related acidosis.
  • Cardiomyocytes were isolated from donor, AS, and DCM hearts. Briefly, samples were defrozen in relaxing solution (in mmol/L: free Mg, 1; KCl, 100; EGTA, 2; Mg-ATP, 4; imidazole, 10; pH 7.0), mechanically disrupted and incubated for 5 min in relaxing solution supplemented with 0.5% Triton X-100. The cell suspension was washed 5 times in relaxing solution. Single cardiomyocytes were selected under an inverted microscope and attached with silicone adhesive between a force transducer and a piezoelectric motor.
  • relaxing solution in mmol/L: free Mg, 1; KCl, 100; EGTA, 2; Mg-ATP, 4; imidazole, 10; pH 7.0
  • Cardiomyocyte F passive was measured in relaxing buffer at room temperature within a sarcomere length (SL) range between 1.8 and 2.4 ⁇ m. Force values were normalized to cardiomyocyte cross-sectional area calculated from the diameter of the cells, assuming a cylindrical shape. As a test of cell viability, each cardiomyocyte was also transferred from relaxing to maximally activating solution (pCa4.5), at which isometric force developed. Once a steady state force was reached, the cell was shortened within 1 ms to 80% of its original length to determine baseline force. Only cells developing active forces >20 kN/m 2 were included in the analysis.
  • SL sarcomere length
  • cardiomyocytes were incubated in relaxing solution supplemented with alkaline phosphatases (AP) (2000 U/mL; New England Biolabs), 6 mmol/L dithiothreitol (MP Biochemicals) for 40min at 20°C and F passive was measured again at SL 1.8-2.4 ⁇ m.
  • AP alkaline phosphatases
  • MP Biochemicals dithiothreitol
  • cardiomyocytes were stretched to ⁇ 2.6 ⁇ m SL, incubated in relaxing buffer with pH 6.6, and held in the stretched state for 15 min (prestretch).
  • cardiomyocytes were returned to slack length and stabilized for 5 minutes before recording the F passive with an identical stretch protocol from SL 1.8-2.4 ⁇ m in the low pH buffer.
  • the pH 6.6 buffer was supplemented with recombinant human ⁇ -B crystallin at 0.1 mg/ml and F passive was measured in the presence of ⁇ -B crystallin again at SL 1.8-2.4 ⁇ m.
  • single AS cardiomyocytes underwent either incubation at pH 6.6 alone or a prestretch in relaxing buffer with pH 6.6 to ⁇ 2.6 ⁇ m SL, followed by the same stretch protocol from SL 1.8-2.4 ⁇ m.
  • F passive was measured in single AS cardiomyocytes from SL 1.8-2.4 ⁇ m before and after incubation with ⁇ -B crystallin, before and after incubation with ⁇ -B crystallin and a prestretch to ⁇ 2.6 ⁇ m SL, and before and after incubation with ⁇ -B crystallin, a prestretch to ⁇ 2.6 ⁇ m SL and incubation at pH 6.6.
  • Frozen human heart tissue was sectioned to a thickness of 5 ⁇ m using a cryostat (Leica). The sections were fixed with 3% paraformaldehyde, permeabilized with 0.05% Tween 20 and immunostained using goat anti- ⁇ -B crystallin (Santa Cruz) diluted 1 in 100 in PBS+1% BSA (immunohistochemical grade; Vector Laboratories). Antigoat conjugated to Alexa 555 (Thermofisher) was used to visualize ⁇ -B crystallin. Membranes were stained using WGA conjugated with Alexa 647 (Thermofisher) diluted 1 in 100 in PBS.
  • Nuclei were visualized using Picogreen reagent (Thermofisher) diluted 1 in 10 000 in PBS. Confocal scanning laser microscopy was performed on a Leica TCS SP8 STED 3X (Leica Microsystems). Picogreen, Alexa 555, and Alexa 647 were irradiated with a pulsed white light laser at 502, 553, and 631 nm, respectively. A 63 ⁇ oil objective with NA 1.4 Numerical Aperture was used to image the sample. Detection of the fluorescent signal was performed with gated Hybrid Detectors. Finally, the images were deconvolved using Huygens Professional (Scientific Volume Imaging).
  • High diastolic stiffness of failing human cardiomyocytes was a significant contributor to overall stiffness of LV myocardial strips of AS and DCM patients ⁇ 20% stretch in AS and ⁇ 25% stretch in DCM.
  • Use of dissected myocardial strips precluded visualization of sarcomeres, and strip lengthening was, therefore, expressed as percentage of stretch with respect to slack length, that is, the minimal length at which Fpassive started to develop ( Figure 1 ).
  • the contribution of cardiomyocyte stiffness to overall stiffness no longer differed between donor and AS cardiomyocytes but continued to differ between donor and DCM cardiomyocytes ( Figure 3 ).
  • Altered cardiomyocyte stiffness can result from isoform shifts of the giant cytoskeletal protein titin, from post-translational modifications of titin such as phosphorylation, formation of disulfide bonds, carbonylation, and s-glutathionylation, or from stretch-induced titin modification. Because of higher expression of the compliant N2BA isoform in AS and DCM, titin isoform shifts do not contribute to the observed rise of cardiomyocyte stiffness observed in AS and DCM cardiomyocytes in the present invention.
  • cardiomyocytes were subjected to a prestretch protocol, which consisted of a 15-minute stretch period at 2.6 ⁇ m followed by a 5-minute stabilization period at slack length.
  • This prestretch protocol was executed in pH 6.6 in donor, AS, and DCM cardiomyocytes.
  • exposure to prestretch caused an upward shift of the diastolic Fpassive-SL relations in AS and DCM cardiomyocytes and a further upward shift of the diastolic Fpassive-SL relation in donor cardiomyocytes ( Figure 4 ).
  • ⁇ -B crystallin protects cardiomyocytes against stretch-induced damage in acidic pH ( Figure 4 ).
  • the present invention also administered ⁇ -B crystallin to AS and DCM cardiomyocytes.
  • ⁇ -B crystallin In contrast to donor cardiomyocytes, ⁇ -B crystallin not only corrected the combined effects of prestretch and acidic pH but also reversed the baseline upward displacement of the diastolic Fpassive-SL relation ( Figure 10 ).
  • This finding was consistent with baseline involvement of previous stretch-induced titin damage in AS and DCM cardiomyocytes and was confirmed in a separate series of experiments, in which ⁇ -B crystallin shifted the diastolic Fpassive-SL relation downward without any previous or concomitant intervention ( Figure 6 ).
  • the magnitude of the downward displacement of the diastolic Fpassive-SL relation was similar in the absence ( Figure 6A ) or presence of foregoing interventions ( Figure 6B and 6C ).
  • the present invention observed upregulation and subsarcolemmal localization of a-B crystallin in AS and DCM cardiomyocytes. Because of the close vicinity of capillaries (white arrows in Figure 7 ), the localization of ⁇ -B crystallin in subsarcolemmal aggresomes was consistent with signals from the microvascular endothelium being involved in their formation. The subsarcolemmal localization also suggested that endogenous ⁇ -B crystallin was diverted from the sarcomeres and therefore failed to exert its protective action on titin distensibility, which was, however, restored after administration of exogenous ⁇ -B crystallin.
  • High cardiomyocyte stiffness significantly contributed to overall myocardial stiffness in AS and DCM. High cardiomyocyte stiffness resulted from titin phosphorylation failing to improve cardiomyocyte stiffness and from previous stretch induced aggregation of titin, both of which were corrected by administration of ⁇ -B crystallin. Diastolic LV dysfunction in heart failure could, therefore, benefit from treatment with ⁇ -B crystallin.

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  1. Zusammensetzung zur Verwendung bei der Behandlung von diastolischer Dysfunktion bei einem Subjekt, wobei die Zusammensetzung eine therapeutisch wirksame Menge einer Substanz umfasst, die den Spiegel und/oder die Aktivität eines kristallinen Proteins in Kardiomyozyten des Subjekts erhöht, wobei das zu behandelnde Subjekt hat: diastolische Herzinsuffizienz; Herzinsuffizienz mit erhaltener Auswurffraktion; Aortenstenose; und/oder dilatative Kardiomyopathie und wobei die Substanz Geranylgeranylaceton (GGA) oder NYK9354 oder ein kristallines Alpha-B-Ketten-Protein der SEQ ID: 1 ist, oder ein Protein, das eine Aminosäuresequenzidentität von wenigstens 80 %, vorzugsweise wenigstens 90 %, bevorzugter wenigstens 95 %, am meisten bevorzugt 98 % oder 99 % mit dem kristallinen Alpha-B-Ketten-Protein hat, wobei das Protein in der Lage ist, eine diastolische Steifigkeit der Kardiomyozyten des Subjekts zu reduzieren.
  2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das kristalline Protein, dessen Spiegel und/oder Aktivität durch die Substanz erhöht wird, ein Protein ist, kodiert durch das kristalline Alpha-B(CRYAB) - Gen.
  3. Zusammensetzung zur Verwendung nach einem der vorstehenden Ansprüche, wobei die Substanz den Spiegel des kristallinen Alpha-B-Ketten-Proteins in den Kardiomyozyten des Subjekts erhöht.
  4. Zusammensetzung zur Verwendung nach Anspruch 3, wobei die Substanz die Expression des CRYAB-Gens in den Kardiomyozyten des Subjekts induziert.
  5. Zusammensetzung zur Verwendung nach Anspruch 1 oder Anspruch 2, wobei die Substanz eine posttranslationale Modifikation der kristallinen Alpha-B-Kette vermittelt, vorzugsweise durch Phosphorylierung des kristallinen Alpha-B-Ketten-Proteins.
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